DK154566B - MONOCLONAL ANTIBODY AND PROCEDURE FOR PREPARING IT - Google Patents

Description

in

DK 154566 B

The present invention relates to a monoclonal antibody and to a method for its preparation.

The fusion of mouse myeloma cells to spleen cells from immunized mice by Kohier and Milstein in 1975 (Nature 256, 495-497 (1975)) demonstrated for the first time that it was possible to obtain a continuous cell line that produces homogeneous (so-called "monoclonal") antibody. Since this basic work, great efforts have been made to produce various hybrid cells (called "hybridomas") and to use the antibody produced by these hybridomas for 10 different scientific studies. For example, reference is made. to Current Topics in Microbiology and Immunology, Volume 81 - "Lymphocyte Hybridomas," F. Melchers, M. Potter, and N. Warner, Editors, Springer-Verlag, 1978, and the references cited therein, C.J. Barnstable, et al.,

Cell, 14, 9-20 (May 1978), P. Parham and W.F. Bodmer, Nature 276, 397-399 (November 1978), Handbook of Experimental Immunology, 3rd Edition, Volume 2, D.M. Wier, Editor, Blackwell, 1978, Chapter 25, and Chemical and Engineering News, January 1, 1979, 15-17. These references simultaneously indicate the rewards and complications of trying to produce monoclonal antibody from hybridomas. Although the general technique is well understood in principle, many difficulties and variations meet for each specific case. In reality, there is no certainty, prior to the attempt to produce a given hybridoma, that the desired hybridoma will be obtained, that if obtained will produce antibody or that the antibody thus produced will have the desired specificity. The degree of success is basically influenced by the antigen type used and the selection technique used to isolate the desired hybridoma.

The targeted preparation of human lymphocyte cell surface antigens monoclonal antibody has been reported only in a few 30 cases. See e.g. Current Topics in Microbiology and Immunology, ibid, 66-69 and 164-169 and below. The antigens used in these reported experiments were cultured human lymphoblastoid leukemia and human chronic lymphocytic leukemia cell lines. Many hybridomas obtained appeared to form antibodies to different antigens on all human 35 cells. None of these hybridomas produced antibody to a predefined class of human lymphocytes.

It should be understood that there are two principal classes of lymphocytes involved in the immune system in humans and animals. The

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The first two of these (the thymus-dependent cell or T-cell) are differentiated into the thymus from hemopoietic stem cells. While in the thymus, the differentiating cells are called "thymocytes". The mature T cells leave the thymus and circulate between the tissues, lymph vessels and bloodstream. These 5 T cells make up a large proportion of the amount of recirculating small lymphocytes. They have immunological specificity and are directly involved in cell-mediated immune responses (such as transplant rejection) as effector cells. Although T cells do not secrete humoral antibodies, they are required during the time of secretion of these antibodies by the second class of lymphocytes mentioned below. Some types of T cells have a regulatory function in other aspects of the immune system. The mechanism of this process of cellular interaction is not yet fully understood.

The second class of lymphocytes (the bone marrow-derived cells or B cells) are those that secrete antibody. They are also formed from hemopoietic stem cells, but their differentiation is not determined by thymus. In birds, they are differentiated into one with a thymus analogous organ, called Bursa Fabricii.

In mammals, however, no equivalent organ has been encountered and it is thought that these B cells differentiate into the bone marrow.

It is now recognized that T cells can be divided into at least several 20 subtypes, called "auxiliary", "suppressor", and "kill" T cells which have the function (respectively) to promote a reaction, suppress a reaction or killing (lighter) foreign cells. These subclasses are well understood within the mouse family, but have only recently been described for human systems. See e.g. RL Evans et al., Journal of Experimental Medicine, Vol. 145, 221232, 1977 and L. Chess and S.F. Schlossman “Functional

Analysis of Distinct Human T-Cell Subsets Bearing Unique Differentiation Antigens ", in" Contemporary Topics in Immunobiology ", 0. Stutman, Editor, Plenum Press, 1977, Volume 7, 363379.

The ability to identify classes or subclasses of T cells 30 is important for the diagnosis of various immunoregulatory anomalies or conditions.

For example. have certain leukemia types and lymphomas different prognosis depending on whether they are of B cell or T cell origin. Thus, the assessment of the disease prognosis depends on the distinction between these two classes of lymphocytes. See e.g. A.C. Aisenberg and J.C. Long, The American Journal of Medicine, 58: 300 (March 1975), D. Belpomme, et al., In "Immunological Diagnosis of Leukemias and Lymphomas", S. Thierfelder, et al., Eds, Springer, Heidelberg, 1977, 33-45, and D. Belpomme, et al.,

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British Journal of Haematology, 1978, 38, 85.

Certain disease states (e.g., juvenile rheumatoid arthritis, malignant conditions, and agammaglobulinemia) are associated with an imbalance of T-cell subclasses. It has been suggested that autoimmune diseases are generally associated with an excess of "assist" T cells or lack of certain "suppressor" T cells, while agammaglobulinemia is associated with an excess of certain "suppressor" T cells. In general, malignant conditions are associated with an excess of "suppressor" T cells.

10 In certain types of leukemia, excess T cells are produced at an inhibited developmental stage. The diagnosis may therefore depend on the possibility of detecting such imbalance or excess. See. for example. J. Kersey, et al., "Surface Markers Define Human Lymphoid Malignancies with Differing Prognoses" in Haematology and Blood Transfusion, Volume 20, Springer-15 Verlag, 1977, 17-24 and the references cited therein.

Acquired agammaglobulemia, which is a disease state in which no immune globulin is produced, comprises at least two different types. In type I, the lack of producing immune globulin is due to an excess of suppressor T cells, whereas in type II it is due to a lack of auxiliary T-20 cells. In both types, there appears to be no defect or defect in the patients' B cells, the lymphocytes responsible for the current secretion of the antibody. However, these B cells are either suppressed or "not assisted", leading to greatly diminished or lack of immuno obul in n production. Thus, the type of acquired agammaglobulinemia 25 can be determined by testing for excess suppressor T cells or the absence of auxiliary T cells.

The identification and suppression of human T-cell classes and subclasses has previously been done using spontaneous autoantibodies or selective antisera for human T-cells obtained by immunizing animals with human T-cells, tapping the animals to obtain serum, and antiserum adsorption. with (e.g.) autologous but not allogeneic B cells to remove antibodies with undesirable reactivities. The preparation of these antisera is extremely difficult, especially in the adsorption and purification steps. The adsorbed and purified antisera 35 may even contain many impurities besides the desired antibody, for several reasons. First, serum contains millions of antibody molecules, even before T cell immunization. Second, the immunization causes antibody formation against a wide range

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4 antigens found on all injected human T cells. There is no selective antibody formation against a single antigen. Third, the titer of specific antibody obtained by such methods is usually very low (e.g., inactive at dilutions greater than 1: 100) and the ratio of g 5 between specific and non-specific antibody is less than 1/10, see e.g. the article cited above by Chess and Schlossman (page 365 et seq.) and the article cited above in the Chemical and Engineering News, which describes the deficiencies of known antisera and the benefits of monoclonal antibody.

One of the T-cell subsets identified by such known antisera has been named TH2 + the subset and has been found to contain both the cytotoxic effector cells for cell-mediated lympholysis and the immunoregulatory suppressor T cells which suppresses both T-cell and B-cell function. This subset contains approx.

20% -30% human peripheral T cells. See e.g. articles by E.L. Reinherz, et al., In J. Immunol. 123: 83 (1979) and New Engl. J. Med. 300: 1061 (1979).

It is known, cf. Proc. Natl. Acad. Sci., USA, vol. 76, no. 8, page 40614065, August 1979, to prepare a monoclonal antibody called OKT4 using a hybridoma cell line. This monoclonal antibody 20KT4 reacts with virtually all normal human peripheral helper T cells.

A new hybridoma cell line has now been found capable of producing novel monoclonal antibody called OKT5 against an antigen found on normal human peripheral cytotoxic and suppressor 25 TH2 + T cells (about 20% of normal human peripheral T cells). The antibody thus formed is monospecific for a single determinant on normal human cytotoxic and suppressor TH2 + T cells and contains essentially no other antihuman immunoglobulin, unlike prior art antisera (which are necessarily contaminated with antibody 30 reactive to numerous human antigens) and to previously known monoclonal antibodies (which are not monospecific for a human cytotoxic / suppressor T cell antigen). Furthermore, this hybridoma can be grown to produce antibody without the need to immunize and kill animals, followed by the trivial adsorption and purification steps necessary to obtain only the impure antisera of the prior art.

A sample of the hybridoma cell line in question was deposited at the American Type Culture Collection, 12301 Parklawn Drive, Rockville,

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5 MD, 20852 on September 18, 1979 and September 28, 1979 and granted respectively. ATCC accession numbers CRL 8013 and CRL 8016.

Accordingly, the monoclonal antibody of the invention of class IgG is peculiar in that it is identical to the 5 monoclonal antibody produced by hybridoma cell line ATCC no.

CRL 8013 or 8016, and that it a) reacts with more than 90% of cytotoxic suppressor THg4 "T cells (about 2030% of all normal human peripheral T cells), but not with normal human peripheral B cells, zero cells or macrophages, b) react with about 80% of normal human thymocytes, c) do not react with THg T cells or OKT4 + T cells, but react with about 68% of OKT4 T cells.

The method of the invention for preparing said monoclonal antibody is characterized by culturing hybridoma cells in a ATCC No. CRL 8013 or 8016 in vitro in a suitable medium or introducing it intraperitoneally into mice and extracting the antibody from the supernatant from in vitro culture or from malignant ascites or mice serum.

The present novel hybridoma cell line ATCC

No. CRL 8013 or 8016 has been prepared using the method of Milstein and Kohier. After immunization of mice with normal human thymocytes, the immunized mice spleen cells were fused with cells of a mouse myeloma line and the resulting hybridomas screened for those with supernatants containing antibody which gave selective binding to normal E-rosified positive human cells. T cells. The desired hybridomas were then cloned and characterized. As a result, the hybridoma cell line ATCC No. CRL 8013 or 8016 was obtained which produces antibody (called OKT5) against an antigen on normal human cytotoxic and suppressor T1 T cells. Not only does this antibody react with normal human peripheral cytotoxic and suppressor THg * T cells, but it also does not react with other normal peripheral blood lymphoid cells, including auxiliary T cells. Furthermore, the cell surface antigen recognized by this antibody is detected in about 80% of normal human thymocytes.

In view of the stated difficulties of the prior art and the lack of success reported using malignant cell lines as antigen, although the hybridoma cell line was obtained in DK 154566 β 6 and known in the art, it was not possible to predict whether it was able to provide the desired hybridoma. It should be emphasized that the unpredictable nature of hybrid cell production does not allow extrapolation from one antigen or cell system to another. It has even been found in connection with the present invention that the use of a T cell malignant cell line as antigen caused the formation of hybridomas which did not form the desired antibody. Attempts to use purified antigens separate from the cell surfaces were also unsuccessful.

The preparation and characterization of the hybrid in question and the resulting antibody will be explained in more detail below.

The method of preparing the subject hybridoma comprises the following steps: 15 A. Mice are immunized with normal human thymocytes.

B. The spleens are removed from the immunized mice and a spleen suspension is prepared in a suitable medium.

C. Suspended spleen cells are fused with mouse myeloma cells from a suitable cell line using a suitable fusion promoter.

D. The mixture of diluted spleen cells, unfused myeloma cells, and fused cells is diluted and cultured in a selective medium which does not nourish the unfused myeloma cells for a time sufficient for the unfused cells to die. .

E. The supernatant in each container (well) containing a hybridoma is evaluated for the presence of antibody to E-rosified positive purified human T cells.

35 F. One selects (for example, by restriction dilution) and clones hybridomas that produce the desired antibody.

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The monoclonal antibody can be produced in one of two ways. The purest monoclonal antibody is formed by in vitro culture of the subject hybridoma in a suitable medium for a suitable period of time followed by recovery of the desired antibody from the supernatant. The appropriate medium 5 and the appropriate culture time are known or can be readily determined. This in vitro method produces substantially monospecific monoclonal antibody that is substantially free of other specific antihuman immunoglobulin. There is a small amount of other immunogiobulin present as the medium contains xenogenic serum (e.g. fetal calf serum). However, this in vitro method cannot produce a sufficient amount or concentration of antibody for some purposes, since the concentration of monoclonal antibody is only about 50 µg / ml.

To produce a much greater concentration of slightly less pure monoclonal antibody, the subject hybridoma may be injected into mice, preferably syngenic or semi-syngeneic mice. The hybridoma will cause antibody-producing tumors to form after a suitable incubation time, which will result in a high concentration of the desired antibody (about 5-20 mg / ml) in the bloodstream and peritoneal exudate (ascites) in the host mice. Although these host mice also have normal antibodies in their blood and ascites, the concentration of these normal antibodies is only approx. 5% of the monoclonal antibody concentration. Furthermore, since these normal antibodies are not antihuman in their specificity, the recovered ascites or monoclonal antibody obtained from serum are substantially free of any contaminating anti-human immunoglobulin. This monoclonal antibody has high titer (active at dilutions of 1: 50,000 or more) and high ratio of specific to non-specific immune globulin (about 1/20). Immunoglobulin produced while incorporating the so-called "k light" myeloma chains are non-specific "nonsense" peptides which simply dilute the monoclonal antibody without reducing its specificity.

Example 1

Preparation of Monoclonal Antibodies 35 A. Immunization and somatic cell hybridization CAF, female mice (Jackson Laboratories, 6-8 weeks old) 17 were intraperitoneally immunized with 2 x 10 10 human thymocytes in 0.2 ml phosphate-buffered saline solution with 14 day intervals. Four days

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8 after the third immunodeficiency, the spleen was removed from the mice and a single cell suspension was prepared by pressing the tissue through a stainless steel mesh.

Cell fusion was performed according to that developed by Kohier and Milstein

ISLAND

5 method. 1x10 splenocytes were fused in 0.5 ml of fusion medium containing 35% polyethylene glycol (PEG 1000) and 5% dimethyl sul foxide in "RPMI 1640" medium (Gibco, Grand Island, NY) with 2 X 10 6 P3X63Ag8Ul myeloma cells delivered by Dr. M. Scharff, Albert Einstein College of Medicine, Bronx. NEW. These myeloma cells secrete IgGj κ light chains.

B. Selection and growth of hybridoma

After cell fusion, cells were grown in HAT medium (hypoxanthine, aminopterin and thymidine) at 37 ° C with 5% COg in a humid atmosphere. Several weeks later, 40 to 100 μΐ supernatant from cultures containing hybridomas were added to a tablet of 106 peripheral lymphocytes separated in E-rosette positive (E +) and E-rosette negative (E ~) populations prepared from blood of healthy human donors. as described by Mendes (J. Immunol. 111: 860, 1973). Detection of mouse hybridoma antibodies that bind to these cells was determined by indirect immunofluorescence. Cells incubated with culture supernatants were stained with fluorescent goat anti-mouse IgG (G / M FITC) (Meloy Laboratories, Springfield, VA, F / p = 2.5) and the fluorescent antibody-coated cells were then analyzed for cytofluorograph "FC200 / 4800A" (Ortho Instruments, Westwood, MA) as described in Example 2. Hybridoma cultures containing antibodies that reacted specifically with E + lymphocytes (T cells) were selected and cloned twice by restriction dilution methods. in the presence of food cells. Then, the clones were transferred intraperitoneally by injection of 1 x 10 6 cells of a given clone (0.2 ml volume) into CAF 2 mice pre-treated with 2,6,10,14-tetramethylpentadecane supplied by Aldrich Chemical Company under the name " Pristine ". The malignant ascites from these mice were then used to characterize lymphocytes as described below in Example 2. The standard hybrid antibody OKT5 was shown by standard technique to be of IgGj subclass.

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Example 2

Characterization of OKT5 Reactivity A. Isolation of Immunocyte DODulations 5 Human peripheral blood mononuclear cells were solubilized from healthy voluntary donors (ages 15-40) by "Ficoll-Hypaque" density gradient centrifugation (Pharmacia Fine Chemicals, Piscataway, NJ) followed by the technique described by Boyum in Scand. J. Cl in.

Lab. Invest. 21 (Suppl. 97): 77, (1968). Unfractionated mononuclear cells are divided into surface Ig + (B) and Ig '(T plus zero) populations by "Sephadex G-200" anti-F (ab') 2 column chromatography as previously described by Chess, et al. , J. Immunol. 113: 1113 (1974). T cells were obtained by E-rosetting the Ig "population with 5% sheep erythrocytes (Microbiological Associates, Bethesda, MD).

The rosetted mixture was layered over Picoll-Hypaque and the obtained E + tablet treated with 0.155M NH 2 Cl (10 ml per 108 cells).

The T-cell population thus obtained was less than 2% EAC rosette positive and more than 95% E rosette positive determined by standard methods.

Furthermore, the non-rosetting Ig "(zero cell) population was recovered from the Ficoll interface. This latter population was less than 5% E + and less than or equal to 2% slg +. The surface Ig + (B) population was obtained from" The Sephadex G-200 "column after elution with normal human gamma globulin as previously described. This population was greater than 95% surface IG + and less than 5% E +.

25 Normal human macrophages were obtained from the mononuclear population by attachment to polystyrene. Thus, mononuclear cells were resuspended in si culture media (RPMI 1640, 2.5 mM HEPES [4- (2-hydroxyethyl) -1-piperazine propane sulfonic acid] buffer, 0.5% sodium bicarbonate, 200 mM L-glutamine and 1% penicillin-streptomycin, 30% added with 20% heat-inactivated human AB serum) at a concentration of 2 x 10 celler cells and incubated in plastic-petri dishes (100 x 20 mm) (Falcon Tissue Culture Dish, Falcon, Oxnard, CA) at 37 ° C overnight. After thorough leaching to remove non-adherent cells, the adherent population was released by copious leaching 35 with cold serum-free medium containing 2.5 mM EDTA and occasional scraping with the rubber tip of the disposable syringe plunger. More than 85% of the cell population was capable of ingesting latex particles and had monocyte morphological properties of

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Wright-Giemsa stain.

B. Isolation of thvmocvter

Normal human thymus gland was obtained from patients with age 5 between 2 months and 14 years who underwent corrective cardiac surgery.

Freshly obtained portions of the thymus gland were immediately placed in 5% fetal calf serum in medium "199" (Gibco) finely parted with forceps and scissors, and then formed into single cell suspensions by pressing through the wire mesh. The cells were then layered over Ficoll-10 Hypaque and spun and washed as described in Section A above.

The thymocytes thus obtained were more than 95% alive and more than or equal to 90% E-rosette positive.

Cell line

An Epstein-Barr Virus (EBV) transformed B-cell line from a normal individual (Laz 156) was provided by Dr. H. Lazarus, Sidney Farber Institute, Boston, MA.

Cytofluoroctrafic analysis and cell division

Cytofluorographic analysis of monoclonal antibodies with all cell populations was performed by indirect immunofluorescence with fluorescein-conjugated goat anti-mouse IgG (G / M FITC) (Meloy Laboratories) using a cytofluorograph "FC200 / 4800A" (Ortho

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25 Instruments). Briefly, 1 x 10 6 cells were treated with 0.15 ml of OCT5 at 1: 500 dilution, incubated at 4 ° C for 30 minutes and washed twice. The cells then reacted with 0.15 ml of a 1:40 dilution G / M FITC at 4 ° C for 30 minutes, centrifuged and washed 3 times. Cells were then analyzed on the cytofluorograph and the fluorescence intensity per cell was recorded on a pulse height analyzer. A similar reactivity pattern was observed at a dilution of 1: 10,000, but further dilution caused loss of reactivity. Background grafting was achieved by substituting a 0.15 ml portion of 1: 500 ascites from a CAF + mouse intraperitoneally injected with a non-producing hybrid clone. Reactivity of lymphoid populations with horse anti-THg and normal horse IgG was determined as previously described in the above articles by Reinherz, et al.

; In experiments designed to separate T cell subsets, 100 x 10 unfractionated T cells were labeled with 4 ml of a 1: 500 dilution of OKT4 or OKT5 and developed with G / M FITC. OCT4 had previously been found to be reactive especially with 55-60% of peripheral blood T-5 lymphocytes representing the human auxiliary subsets. Using fluorescence-activated cell sorter (FACS-I) (Becton-Dickinson, Mountain View, CA), T cells were subdivided into 0KT4 + and 0KT4 "subsets as well as 0KT5 + and OKT5 'subsets from the same individual. more than 95% by Trypan Blue 10 exclusion in all cases The purity of all separated populations was greater than or equal to 95%.

Analysis of anti-TlL equine T-cell submenuts

In the same way as above, horses used anti-TH «. * g 15 separation of TH2 and TH2 ~ T cells on FACS by labeling 60 x 10 unfractionated T cells with 0.12 ml equine anti-TH2 and 0.1 ml R / H FITC (Cappel Laboratories, Downington, PA) as previously described by Reinherz, et al. Purity and viability of sorted cells were similar to the sorted populations described above. FACS-sorted T cell 20 aliquots isolated with equine anti-TH2, OKT4 or OKT5 were cultured for 48 hours at 37 ° C in 5% CO 2 moist atmosphere with RPMI 1640 containing 20% human AB serum, 1% penicillin streptomycin , 200 mM L-glutamine, 25 mM HEPES buffer (Microbiological Associates) and 0.5% sodium bicarbonate. These cultured cells were then analyzed as described above on the Cytofluorograph. Background staining was determined by substituting normal horse IgG for specific antibody and staining as above.

Functional Studies 30 A. Formerinas Studies c

The mixed reaction of 10 unspecified and FACS-fractionated T lymphocytes was tested in microculture for optimal doses of Concanava lin A (Con A) (Calbiochem, La Jolla, CA) and phytohemagglutinin (PHA) 35 (Burroghs-Wellcome Company, Greenville , NC). Alloantigen proliferate response was simultaneously measured for these same populations with mitomycin-treated Laz 156, an EBV-transformed human B lymphoblastoid cell line stimulus. Tetanus Toxoid Proliferation (Massachusetts

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The Department of Public Health Biological Laboratories, Boston, MA) and Sheep Disease Antigen (Microbiological Associates) were tested using a 10 µg / ml final concentration and a 1:20 dilution. 5% macrophages obtained in the manner described above were added to all 5 populations at the initiation of in vitro cultures. Mitogen-stimulated 3 cultures were pulsed after 4 days with 0.2 µCi of H-thymidine (H-TdR; 1.9 Ci / mM specific activity) (Schwarz-Mann, Division of Becton,

Dickinson, Orangeburg, NY) and selected 18 hours later on a "MASH Π" 3 apparatus (Microbiological Associates). H-TdR incorporation was measured on a Packard Scintillation Counter (Packard Instrument Company, Downer's 3).

Grove, IL). Background H-TdR incorporation was obtained by substituting medium for mitogen. Soluble and cell-surface alloantigen cultures were pulsed after 5 days with H-TdR for 18 hours, diluted and counted as described above.

15 B. Cytotoxic studies

Sensitization cultures for cell-mediated lympholysis (CML) were established by placing unfractionated T cells with mitomycin-treated stimulator cells, all at 2 x 10 6 cells per ml, in a number of 20 microtiter plate wells. After 5 days, the unfractionated T cells were fractionated into OKT5 + and OKT5 ”T cell subsets on FACS.

These T-cell subsets were then added to ® * Cr sodium chromate-labeled attack cells and specific cytotoxicity determined after 6 hours of cell incubation. The percent cytotoxicity was determined by the following formula: 51 51

Cr free on trial - Cr released spontaneously 51Cr released by freeze-drying - 51Cr released spontaneously X 100 30 All samples were run in triplicate and the results expressed as mean ± standard deviation.

C. Con A activation of suppressor cells and suppression of MLC

Unfractionated T cells were activated with 20 µg Concanavalin A

35 (Con A) (Calbiochem) per 10® cells. These Con A treated cells 2

cultured in tissue culture flasks with a surface area of 25 cm (Falcon, Oxnard, CA) in RPMI 1640 (Grand Island Biological Company) containing 20% human AB serum, 1% penicillin-streptomycin, 200 mM

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13 L-glutamine, 25 mM HEPES buffer (Microbiological Associates) and 0.5% sodium bicarbonate for 48 hours at 37 ° C in a humid atmosphere containing 5% CO 2. Untreated control T cells were cultured identically, but without Con A. Next, the cells were centrifuged, washed five times and added to fresh autologous responder cells in one-way mixed lymphocyte culture (MLC) as described above. In suppression experiments, one-way MLCs were obtained in mi crotiter plates with round bottom (Linbro Chemical Company,

New Haven, CT) and triple indentations, each containing 0.05 x fi 10 responder lymphocytes (whole mononuclear), 0.05 x 10 either 10 unactivated or Con A activated autologous unfractionated or

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fractionated T cells and 0.1 x 10 10 mitomycin-treated stimulated cells (Laz 156). After five days, cultures were pulsed with 0.2 μΟι 3 of H-TdR and diluted after 18 hours as described above. Percent inhibition of MLC propagation was then calculated using the formula: + inhibition% = (1- CR - - A) x 100 cpm 20 wherein cpm Con A + denotes the results of 1 H-TdR incorporation into MLC when added Con A activated autologous T cells or T ~ cell subsets, and wherein cpm denotes results when unactivated autologous T cells are added.

The drawing is referred to below.

Figure 1 shows the fluorescence pattern obtained on the cytofluorograph after reaction of normal human, peripheral T cells, B cells, null cells and macrophages with OKT5 at 1: 1000 dilution and G / M F1TC in the upper row. For comparison, results with horses anti-TH2 are shown in the lower row.

Figure 2 shows the fluorescence pattern obtained on the cytofluorograph after reaction of human thymocytes with OKT5 and G / M FITC (A) and with horses anti-TH2 (B).

Figure 3 shows the fluorescence pattern obtained on the cytofluorograph after reaction of horses anti-TH2 separated T-cell amounts with OKT5.

Figure 4 shows the fluorescence pattern obtained on the cytofluorographer · after reaction of 0KT4 separated T cell subsets with 0KT5.

Figure 5 shows the cytotoxic capacity of unfractionated T cells and T cell subsets separated by OKT5 after all o-sensitization in

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14 MLC.

As shown in the upper row of Figure 1, approximately 20% of the human peripheral blood T cell population of a given normal subject is reactive with OKT5, while the entire amount of B cell, zero cell and macrophage populations are isolated. from the same individual are unreactive with 0KT5. Similarly, horses react anti-THg with 24% of peripheral T cells and are also unreactive with B cells, zero cells and macrophages. The monoclonal antibody is therefore characterized in that it is reactive with an antigen contained on the surface of ca. 20% of normal human peripheral T cells, 10 but unreactive with all antigens on the surface of the other three cell types mentioned above. As will be discussed below, the OKT5 + proportion of the human peripheral T cell is included in the cytotoxic and suppressor T cell subset. This differential reactivity constitutes one test by which the subject antibody OKT5 can be detected and distinguished from other antibodies.

As shown in Figure 2, about 80% of normal human thymocytes from a 6-month-old child are reactive with OKT5. Similar results (approx.

80% reactivity) was obtained using additional thymus samples from normal individuals aged 2 months to 19 years. This value is 20 the same as for horses anti-Tl · ^. The reactivity pattern of Figure 2 provides another method for detecting the subject antibody OKT5 and for distinguishing it from other antibodies.

As shown in Figure 3, the antibody in question reacts with TH2 + but not with TH2 "T cells. Approximately 5-10% of the TH2 + subset did not react with 25 OCT5. The reactivity pattern of Figure 3 provides a third method for detecting the antibody in question. 0KT5 and to distinguish it from other antibodies.

As shown in Figure 4, the OKT4 + T cell subset does not react with OKT5 at all. In contrast, the 0KT4 + T cell subset is essentially 0KT5 + (6,800 out of 10,000 cells tested). These results show that, like the previously determined TH2 + subset, the 0KT5 + subset is reciprocal and different from the 0KT4 + subset. While the OKT4 + subset contains the helper T cells, the OKT5 + subset (like the TH2 + subset) contains cytotoxic and suppressor T cells.

This pattern of reactivity in Figure 4 provides a further method for the determination of OKT5 antibody and to distinguish it from other antibodies.

Figure 5 shows that the OKT5 + T cell subset influences CML.

The degree of lysis mediated by this population is greater than that,

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15 which is mediated by the unfractionated T cell population. In contrast, the OKT5 "T cell population is minimally lytic. After activation in MLC against Laz 156, the unfractionated T cells were separated into OKT5 + and OKT5" subsets. Both unfractionated and fractionated T cells 51 were analyzed in CML against Cr labeled Laz 156 targets at different effector: target (E: T) ratios. The OKT5 + T cell subsets contained the effector population in cell-mediated lympholysis. At an E: T ratio of 5: 1, 10: 1 and 20: 1, the OKT5 + T cell population, respectively, provided. 40%, 58% and 77% specific lysis. The lytic capacity of the isolated OKT5 + 10 subset was significantly higher than the unfractionated T cell population. In addition, the 0KT5 “T cell population provided significantly less lysis at the E: T ratios tested. Looking at the previous observation that the TH2 + T cell subset in humans provides CML, the present results will support the assumption that the 15 TH2 + and OKT5 + T cell subsets define similar populations of functionally active T lymphocytes. This differential cytotoxic capacity provides yet another method for the determination of OKT5 antibody and to distinguish it from other antibodies.

Functional studies were performed on lymphoid populations that had been separated on a fluorescence-activated cell separator (FACS).

The results of these studies are shown in Tables (I) to (III) below and provide further basis for the previously described characterization of the monoclonal antibody in question.

In these studies, an unfractionated T cell population 25 was treated with a 1: 500 dilution of OKT5 and G / M FITC and separated on FACS in OKT5 + and OKT5 "subsets. With the purity of the populations obtained given (greater than or equal to 95% ), 5% macrophages were added to the separated populations before culturing in vitro.The unfractionated T-cell iI population and the isolated OKT5 and OKT5 T-cell subsets were then stimulated with PHA, Con A, soluble antigens and allo antigens for determination. of their in vitro propagation reactions.

The propagation response of the unfractionated T cell populations on PHA and Con A is shown in Table (I). A maximal propagation reaction g in the unfractionated T cell population is achieved with 1 µg per 10 cells with diminished responses at 0.5 µg and 0.1 µg PHA per g 10 cells. Treatment of the unfractionated T cells with OKT5 and goat mouse FITC without subsequent fractionation did not alter the propagation reaction. In contrast, differences in reaction rates were obtained.

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16 on the PHA with the separated OKT5 + and OKT5 'T cell subsets. The OKT5 ”population responded to all doses of PHA in a manner similar to the unspecified T cell population. However, the propagation response of OKT5 + cells was significantly less at all doses of PHA tested. In addition, the OKT5 + T cells did not propagate at all at a PHA dose of 0.1 µg per 10 cells, whereas the OKT5 + T cell subset and the unfractionated cells still reacted. However, the propagation response of these subunits on Con A was similar and the two subsets of cells could not be distinguished from each other or from the unfractionated T cell 10 population.

The reactions to all the faces in MLC and to soluble antigens were then investigated. As shown in Table (II), the unfractionated T cell population, the non-fractionated T cell population treated with 0KT5 and G / M FITC, and both the 0KT5 + and 0KT5 'T cell subsets reacted similarly in MLC against Laz 156. In contrast, proliferative responses to soluble antigens provided the clearest distinction between these subsets. In all cases investigated, the OKT5 + T cell subset proliferated minimally against soluble antigens, tetanus toxoid and mumps, whereas the OKT5 + T cell subset responded well.

20 Previous studies showed that the TH2 + population of T lymphocytes could be induced with Con A to suppress autologous lymphocytes in MLC.

To determine if 0KT5 + T cell subsets could similarly express suppressor function, T cells were activated for 48 hours with Con A and then sorted with 0KT5 and OCT4 monoclonal antibody into specific 25 T cell subsets. Next, these separated T-cell subpopulations were added to autologous responding lymphocytes at the onset of MLC. As shown in Table III, with Con A, unfractionated T cells suppressed autologous cell proliferation in MLC. Treatment with monoclonal antibody and G / M FITC did not change this result. Although both OCT4 + and OCT5 + T cells multiplied with Con A, only OCT5 + T cells were suppressive when activated with Con A. The OCT4 + subset did not suppress. In addition, the 0KT5 subpopulation, which contains both the 0KT4 + and 0KT4 "TH2 ~ subpopulations, appeared minimally oppressive when compared to the highly suppressive 0KT5 + 35 population.

Table (IV) shows the association between the level of peripheral T cells and T cell subsets and different disease states. This context can be used for diagnostic purposes (eg to detect acute infectious)

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17 mononucleosis) by analyzing the blood sample of an individual suspected of having one of these disease states to determine the level of T-cells and T-cell subsets. The relationships shown in Table (IV) are another way in which OKT5 antibody can be detected and distinguished from other antibodies.

18 DK 154566 B

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21

Table IV

Peripheral T-cell levels at disease states

Disease condition 0KT3 + 0KT4 + 0KT5 +

Primary biliary cirrhosis (2) N + 5 Disseminated sclerosis (advanced disease) (8) - N -

Myasthenia Gravis (incipient, untreated) (3) 000

Acute transpiration / host (3) 0 to - 0 10 Acquired agammaglobulinemia type I + type II 0

Hyper-IgE (4) - N 0 to - *

Acute infectious mononucleosis (4) + 0 to - ++ 15 Hodgkin's disease

I. and II. stage N N N

III. and IV. stage N N

N = within normal limits 20 0 = absent + = above normal ++ = much above normal - = below normal - = much below normal 25 these levels return to normal approx. a week before the clinical symptoms disappear "The numbers in brackets indicate the number of patients assessed." 30

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22

It was shown that the antibody in question OKT5 belongs to the IgGβ subclass which is one of the four mouse IgG subclasses. These subclasses of immunoglobulin G differ in the so-called "fixed" regions, however, an antibody for a specific antigen will have a so-called "variable" region which is functionally identical regardless of which subclass of immunoglobulin G it belongs. The antibody in question can be used to detect too high cytotoxic or suppressor T-cell activity by reacting a T-cell preparation from a subject with OKT5 antibody. Excessive cytotoxic or suppressor T cell 10 activity will be detected in the presence of more than 20-30% of the total peripheral T cell population that reacts with OKT5. This diagnostic technique can be used using OKT5 antibody alone or in combination with other antibodies (eg OKT3 and OKT4) as shown in Table IV. Reactivity patterns with a selection of antibodies to T cells 15 and T cell subsets allow more accurate detection of certain disease states than would be possible by prior diagnostic methods.

Claims (2)

1. IgG monoclonal antibody, characterized in that it is identical to the monoclonal antibody produced by the hybridoma cell line ATCC No. CRL 8013 or 8016, and that it (5) reacts with more than 90% of cytotoxic and suppressor TH2 + T cells (about 20-30% of all normal human peripheral T cells), but not with normal human peripheral B cells, null cells, or macrophages; 80% of normal human thymocytes; c) do not react with TH2 'T cells or OKT4 + T cells, but react with ca. 68% of OKT4 "T cells. A method of producing the monoclonal antibody of claim 1, characterized in that the hybridoma cell line ATCC No. CRL 8013 or 8016 is cultured in a suitable medium or introduced into mouse intraperitoneally and extracted from the in vitro culture supernatant or from malignant ascites or serum from the mice. 20 25 30 35